Contracting the Host Range of Bacteriophage T7 Using a Continuous Evolution System.

Bacteriophage T7 is an intracellular virus that recognizes its host via tail and tail fiber proteins known as receptor-binding proteins (RBPs). The RBPs attach to a specific lipopolysaccharide (LPS) displayed on the host. While there are various reports of phage host range expansion resulting from mutations in the RBP encoding genes, there is little evidence for contraction of host range. Notably, most experimental systems have not monitored changes in host range in the presence of several hosts simultaneously. Here, we use a continuous evolution system to show that T7 phages grown in the presence of five restrictive strains and one permissive host, each with a different LPS, gradually cease to recognize the restrictive strains. Remarkably, this result was obtained in experiments with six different permissive hosts. The altered specificity is due to mutations in the RBPs as determined by gene sequencing. The results of using this system demonstrate a major role for RBPs in restricting the range of futile infections, and this process can be harnessed to reduce the host range in applications such as recognition and elimination of a specific bacterial serotype by bacteriophages.

Inhibition of host cell division by T5 protein 008 (Hdi).

IMPORTANCE: We have identified a novel phage-encoded inhibitor of the major cytoskeletal protein in bacterial division, FtsZ. The inhibition is shown to confer T5 bacteriophage with a growth advantage in dividing hosts. Our studies demonstrate a strategy in bacteriophages to maximize their progeny number by inhibiting escape of one of the daughter cells of an infected bacterium. They further emphasize that FtsZ is a natural target for bacterial growth inhibition.

Highly active CRISPR-adaptation proteins revealed by a robust enrichment technology.

Natural prokaryotic defense via the CRISPR-Cas system requires spacer integration into the CRISPR array in a process called adaptation. To search for adaptation proteins with enhanced capabilities, we established a robust perpetual DNA packaging and transfer (PeDPaT) system that uses a strain of T7 phage to package plasmids and transfer them without killing the host, and then uses a different strain of T7 phage to repeat the cycle. We used PeDPaT to identify better adaptation proteins-Cas1 and Cas2-by enriching mutants that provide higher adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation in vivo. In vitro, one mutant has higher integration and DNA binding activities, and another has a higher disintegration activity compared to the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.

Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains.

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.

A phage mechanism for selective nicking of dUMP-containing DNA.

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence.

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction-modification systems, is also active against BREX. In contrast to the wild-type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R-M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.

A continuous evolution system for contracting the host range of bacteriophage T7.

Bacteriophage T7 is an intracellular parasite that recognizes its host via its tail and tail fiber proteins, known as receptor-binding proteins (RBPs). The RBPs attach to specific lipopolysaccharide (LPS) features on the host. Various studies have shown expansion of the phage's host range via mutations in the genes encoding the RBPs, whereas only a few have shown contraction of its host range. Furthermore, most experimental systems have not monitored the alteration of host range in the presence of several hosts simultaneously. Here we show that T7 phage grown in the presence of five restrictive strains and one permissive host, each with a different LPS form, gradually avoids recognition of the restrictive strains. Remarkably, avoidance of the restrictive strains was repeated in different experiments using six different permissive hosts. The evolved phages carried mutations that changed their specificity, as determined by sequencing of the genes encoding the RBPs. This system demonstrates a major role for RBPs in narrowing the range of futile infections. The system can be harnessed for host-range contraction in applications such as detection or elimination of a specific bacterial serotype by bacteriophages.

A genetic system for biasing the sex ratio in mice.

Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.

Optimizing DNA transduction by selection of mutations that evade bacterial defense systems.

We recently developed a platform where phage-transducing particles optimize DNA delivery to a wide range of hosts. Here, we use this platform to optimize DNA transduction into hosts that naturally restrict specific DNA sequences. We first show that a specific plasmid is restricted for transduction into a particular Salmonella strain. Using the platform, we select for a mutated plasmid that overcomes the restriction barrier. Insertion of the non-mutated sequence into a permissive plasmid restricts transduction. We further show that epigenetic modification enables the DNA to evade restriction by the putative defense system. Our results validate this straightforward genetic approach for optimization of DNA transduction into new hosts.

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.

Extending the Host Range of Bacteriophage Particles for DNA Transduction.

A major limitation in using bacteriophage-based applications is their narrow host range. Approaches for extending the host range have focused primarily on lytic phages in hosts supporting their propagation rather than approaches for extending the ability of DNA transduction into phage-restrictive hosts. To extend the host range of T7 phage for DNA transduction, we have designed hybrid particles displaying various phage tail/tail fiber proteins. These modular particles were programmed to package and transduce DNA into hosts that restrict T7 phage propagation. We have also developed an innovative generalizable platform that considerably enhances DNA transfer into new hosts by artificially selecting tails that efficiently transduce DNA. In addition, we have demonstrated that the hybrid particles transduce desired DNA into desired hosts. This study thus critically extends and improves the ability of the particles to transduce DNA into novel phage-restrictive hosts, providing a platform for myriad applications that require this ability.

Sensitizing pathogens to antibiotics using the CRISPR-Cas system.

The extensive use of antibiotics over the last century has resulted in a significant artificial selection pressure for antibiotic-resistant pathogens to evolve. Various strategies to fight these pathogens have been introduced including new antibiotics, naturally-derived enzymes/peptides that specifically target pathogens and bacteriophages that lyse these pathogens. A new tool has recently been introduced in the fight against drug-resistant pathogens-the prokaryotic defense mechanism-clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) system. The CRISPR-Cas system acts as a nuclease that can be guided to cleave any target DNA, allowing sophisticated, yet feasible, manipulations of pathogens. Here, we review pioneering studies that use the CRISPR-Cas system to specifically edit bacterial populations, eliminate their resistance genes and combine these two strategies in order to produce an artificial selection pressure for antibiotic-sensitive pathogens. We suggest that intelligent design of this system, along with efficient delivery tools into pathogens, may significantly reduce the threat of antibiotic-resistant pathogens.

Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model.

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

Counteracting selection for antibiotic-resistant bacteria.

The occurrence of antibiotic-resistant bacterial pathogens is on the rise because antibiotics exert selection pressure that kills only the antibiotic-sensitive pathogens. Sanitation and cleansing of hospital surfaces and the skin of medical personnel do not counteract this selective pressure, but rather indiscriminately reduce total pathogens on treated surfaces. Here, we discuss two recently introduced genetic strategies, based on temperate bacteriophages as DNA-delivery vehicles, that aim to sensitize bacteria to antibiotics and selectively kill the antibiotic-resistant ones. Outlooks for rendering one such approach more efficient and applicable are proposed. We believe that using an end product designed according to the provided principles on hospital surfaces and in hand-sanitizers will facilitate substitution of antibiotic-resistant pathogens with sensitive ones.

Phenotypic heterogeneity in a bacteriophage population only appears as stress-induced mutagenesis.

Stress-induced mutagenesis has been studied in cancer cells, yeast, bacteria, and archaea, but not in viruses. In a recent publication, we present a bacteriophage model showing an apparent stress-induced mutagenesis. We show that the stress does not drive the mutagenesis, but only selects the fittest mutants. The mechanism underlying the observed phenomenon is a phenotypic heterogeneity that resembles persistence of the viral population. The new findings, the background for the ongoing debate on stress-induced mutagenesis, and the phenotypic heterogeneity underlying a novel phage infection strategy are discussed in this short manuscript.

Programming Bacteriophages by Swapping Their Specificity Determinants.

Bacteriophages, bacteria's natural enemies, may serve as potent antibacterial agents. Their specificity for certain bacterial sub-species limits their effectiveness, but allows selective targeting of bacteria. Lu and colleagues present a platform for such targeting through alteration of bacteriophages' host specificity by swapping specificity domains in their host-recognition ligand.

Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria.

The increasing threat of pathogen resistance to antibiotics requires the development of novel antimicrobial strategies. Here we present a proof of concept for a genetic strategy that aims to sensitize bacteria to antibiotics and selectively kill antibiotic-resistant bacteria. We use temperate phages to deliver a functional clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system into the genome of antibiotic-resistant bacteria. The delivered CRISPR-Cas system destroys both antibiotic resistance-conferring plasmids and genetically modified lytic phages. This linkage between antibiotic sensitization and protection from lytic phages is a key feature of the strategy. It allows programming of lytic phages to kill only antibiotic-resistant bacteria while protecting antibiotic-sensitized bacteria. Phages designed according to this strategy may be used on hospital surfaces and hand sanitizers to facilitate replacement of antibiotic-resistant pathogens with sensitive ones.

Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) comprise a prokaryotic adaptive defense system against foreign nucleic acids. This defense is mediated by Cas proteins, which are guided by sequences flanked by the repeats, called spacers, to target nucleic acids. Spacers designed against the prokaryotic self chromosome are lethal to the prokaryotic cell. This self-killing of the bacterium by its own CRISPR-Cas system can be used to positively select genes that participate in this killing, as their absence will result in viable cells. Here we describe a positive selection assay that uses this feature to identify E. coli mutants encoding an inactive CRISPR-Cas system. The procedure includes establishment of an assay that detects this self-killing, generation of transposon insertion mutants in random genes, and selection of viable mutants, suspected as required for this lethal activity. This procedure enabled us to identify a novel gene, htpG, that is required for the activity of the CRISPR-Cas system. The procedures described here can be adjusted to various organisms to identify genes required for their CRISPR-Cas activity.

CRISPR adaptation biases explain preference for acquisition of foreign DNA.

CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.